X-Message-Number: 4986
Date: 14 Oct 95 16:53:36 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS:Response to Brad

I thought Brad Templeton's questions about cold ischemia and synapse 
preservation were very good ones; I did not respond before because I wanted 
to see what Brian would say.

I will try to address Brad's concerns:

First, we have done experiments with 24 hours of flush-store on ice using 
dogs with NO warm ischemia and ideal treatment wherein they were put on 
bypass while alive, colled to 15 C and washed out and further cooled to 4C 
and then packed in ice (1-2 C) using both our own solution and Viaspan. 
This was followed by cold perfusion fixation, and TEM microscopy.  Both 
solutions gave good preservation of synapses.  However, to our surprise 
(and mixed pleasure/displeasure) the brains using our solution looked much 
better than the Viaspan brains.  The principal differences were massive 
swelling of the axons (particularly the myleinated ones) and swelling of 
the mitochondria with lost of cristae in many neurons and glial cells in 
the Viaspan group.  Our solution was indistinguishable from control 
(vitally perfused-fixed dogs) at 24 hours.

Brad goes on to say:

>I don't see how you could detect high-level memory loss easily in dogs.
>What level of testing was done?

Brian responds to this question by pointing out that dogs recovered from 
TBW still know their names and exhibit normal kennel behavior.

This is nice to know, but it does not address Brad'd question, which is a 
VERY good one.  Before I address it I will jump to a seemingly unrelated 
topic:

In the last issue of Alcor's magazine CRYONICS there is an article by 
Thomas Donaldson on conservation of memory after ischemic insult.  I had 
high hopes for this article but was very disappointed upon reading it.  One 
thing which I have seen repeatedly cited by BOTH Donaldson and Merkle (AND 
for the same reasons!!!!!) is the story of Hossman's one hour ischemic cat 
which survived and recovered (walked around).  This story is relevant 
because of it denouement and BECAUSE it bears directly on Brad's question.

What about this marvelous cat??  What should we make of it?

1) Not much since only ONE cat survived and no one has come even close to 
duplicating this work.

2) Does anyone know what happened to this SPECIFIC margic cat?  I do.  It 
is an interesting story.  Scientists are heartless souls and German 
scientists probably even more heartless (note I said heartless not 
malicious).  The cat in question was allowed to wander around Hossman's lab 
for some months where it rubbed against people and generally behave like a 
cat.  Which is to say it slept a lot, ate when it wanted and was the 
typical snotty cat like the one sitting on my lap (cats do, after all, look 
down on people).

Finally they decided the cat's time had come and they anesthetized it and 
perfused it with fixative. They then looked at various brain areas.  There 
were heavy neuron losses across the board.  There were, and I quoting 
Hossman here, NO visible hippocampal neurons; just open space and scarring 
(fibrous tissue).

There is a gruesome but relevant experiment you can do with mice.  Mice 
have very thin skulls and you can see the brain through them.  If you 
reflect the skin over a mouse's brain and repeatedly over a period of weeks 
progressively freeze the cerebral cortex through the bone you will 
eventually end up with a mouse with no upper cortical layers and just a 
series of sort of grayish empty pits visible under the bone.  Such mice do 
quite well; they eat, run around and have sex.  However, you cannot TEACH 
them anything.  They are just little reptilian brains.  As I sit here I am 
watching two large red-eared slider turtles.  

I just fed them and they are eating their Purina trout chow; they are 
incredibly stupid animals (but I love 'em anyway) and have barely learned, 
after all this time (a year) that the food is NOT where the motion is; they 
run up on the rocks where they see me, and then it takes them a long while 
to realize I have put the food in at the water end of the tank (this not 
cruelty or devilment; sliders cannot eat food except in the water; they 
would starve if I put it on the rocks).

Brad is very perceptive: assessing neurological injury and in particular 
(and of most relevance to US) cognitive injury in dogs or other animals is 
not easy. I am moving beyond Peter Safar's overall performance catergories 
to much more sophisticated measures of canine intelligence and cognition.  
There is an excellent, indeed truly wonderful book that was published 
recently which describes objective ways to evaluate cognitive function in 
dogs.  Further, we have developed sophisticated techniques of our own to do 
functional neurologicalevaluation.

This is a VERY important and tricky business.  BPI currently has a client 
who is dying.  S/he is quite a brilliant person and has a mind that 
operates scattershot like a machine gun; jumping from subject to subject 
with lightning speed.  I have called in as consults several of the best 
physicians in the United States to deal with various aspects of managing 
this patient's care.  One consult flew in from the East Coast.  One of the 
things I noticed about this patient was his/her noncompliance and seeming 
inability to follow directions.  It took me an actual MEETING with the 
person before I realized what was wrong: s/he had profound cognitive 
impairment; almost no short-term memory.  While the person was witty, 
appropriate, fully oriented to person, place and time, and able to give 
complex instructions to get me to an eatery for lunch, s/he was unable to 
remember a number given when we paused during our wide ranging conversation 
in the following manner:

"Mr/Ms X, I want to interrupt our conversation here.  This is very 
important.  I am going to ask you to remember a number in order to see how 
good your memory for new things is.  The number is 453.  Can you remember 
that?"  The MD got an irritated "of course!" as a response.  Five minutes 
later he stopped the conversation and asked for the number; the person 
could only NOT give the MD the number, indeed s/he had some initial 
puzzlement as to what he was aking for!  Because of the thinking style of 
this individual (very nonlinear and nontechnical) this devastating loss of 
short term memory was almost invisible.  This person was not and is not 
demented and does not have Alzheimer's, but rather is suffering from a 
serious but fairly compartmentalized cognitive defect.  It was of great 
clinical significance to identify this defect since it was preventing 
medical compliance, preventing intake of premeds, resulting in overdoses of 
other prescribed meds (didn't remember taking the pills, took MORE) and 
most importantly was resulting in severe malnutrition since this person was 
preparing his/her own meals!

To belabor the point further, Wilder Penfield (the father of modern 
neurosurgery) had a patient with severe BILATERAL temporal lobe epilepsy 
which was uncontrolled by drugs.  Penfield operated on monkeys ablating 
BOTH of the areas in the temporal lobes he intended to ablate in this 
patient.  The monkeys did fine;  they remembered everything they had been 
taught and appeared normal.  Penfield then operated on his young human 
patient.  Everything went swimingly well!  The patient woke up, the 
seizures were gone, and he knew everybody.  The problem was detected a few 
dys later when it became apparent that he had essentially NO short-term 
memory.  If he met someone for the first time and they walked out of the 
room to return 10 minutes later he had no idea who they were.  

As of some years ago this unfortunate man was living in the same house he 
had since childhood with the same issues of Reader's Digest sitting around, 
the same furniture in the same place.  If he goes outside and sees new cars 
and jet planes he becomes excited and confused.  He is thus marooned in the 
mid-1950's.  

When Penfield went back and checked his monkeys, he found they too were 
marooned in the past. It just wasn't obvious.

We have become VERY sensitive to this issue in dogs in our TBW and ischemia 
experiments.  Dogs that LOOK just fine will sometimes have almost no 
neurons left in multiple areas of their brains.  This work ain't simple!

>Clearly the answer is simple, to examine perfused tissues in animals after
>a long cold ischemia period matching the pattern of a typical suspension,
>look at the detailed neural structure, see how much it has degraded and
>speculate on whether it can be repaired, and how to improve it.

Again Brad is right on the money.  As I said, we have done work on 
nonshocked dogs after 24 hours cold ischemia.  But what we need to do is 
shock/global ischemia models as well, which paralell the long agonal 
courses of patients with SYSTOLIC blood pressures of 50 mmHg or BELOW who 
lay there with pupils midposition and unresponsive to light for 1-2 days in 
this condition before dying!  If you doubt me on this, or how common it is, 
I suggest you call up your local home-hospice or freestanding in-patient 
hospice and talk to a few of the nurses there.  They'll set you straight in 
a hurry.

>How much does such work cost?  I presume it requires electron microscopy.

Yes, currently we are doing EM.  But this is hardly the whole picture.  
There is a new form of vital light microscopy which one of our 
investigators flew out to DuPont to see.  Very impressive: EM resolution 
without artifact on living tissue.  The hardware is exotic and out of our 
price range, but we are working on other ways to get access to this 
technology. Further, there are lovely probes that can be used to image and 
evaluate brain tissue function in slices and in whole brains such as 
voltage sensitive dyes and "functional connection mapping using 
intracelluar electrodes....  

Also, freeze fracture (a kind of EM) could probably tell us a lot.  But we 
have had real problems here getting images of CPA-loaded systems which are 
interpretable.  Again, time and money have limited me in following this up.

Unlike CI who rely on workers overseas, I consider the details of EM 
prep-work and execution to be critically important to getting good results. 
 I fly up initially when the first batch is run, and I fly up when the 
tissue is scanned.   I have tried "cheap" EM services.  You get what you 
pay for.  Excluding the animal, surgical, disposables, chemicals and other 
prep costs to simulate the typical human cryopreservation, EM is 
comparatively cheap.  We work up 3 blocks from each of 12 brain areas on a 
full survey and get an average of 6-12 pictures per block.  This is a LOT 
of EM!!!!!!!  Cost for such a complete work-up are in the 4-6K range 
depending on how many different magnifications I want.  I think this is a 
very good price, especially considering they have to deal with moi!

The cost (marginal) to fully simulate a human cryopresevation (excluding 
prolonged shock) is about 3K to 5K depeding on personnel availability, and 
so on (some folks work for free; actually the noncryonicists are some of my 
best volunteers, not that there aren't as many or more committed 
cryonicists as volunteers).  I have NOT done prolonged shock models due to 
the cost and the certain knowledge that I will have to run through some 
animals to get the model worked out.  If anyone is willing to pay for such 
work, I'm willing to do it, indeed anxious to.  Right now, it is not so 
much of a priority for me because I am focused on solving the big 
underlying problem which is at the root of WHY so many patients end up 
experiencing this kind of insult: improper or inadequate pre-arrest 
pharmacologic cerebral protection, development of appropriate reperfusion 
techniques after global and trickle-flow ischemic injury, and finally, 
reversible brain cryopreservation.  

There is only so much time (and money) I can spend on survey work (such as 
finding the answers to Brad's good questions and the many variants that 
spring fom them such as: well are synapses intact after 2 hours of global 
ischemia followed by washout?  What is the effect of a prolonged, 
unsuccessful "code" on ultrastructure after TBW, etc.).  I have to decide 
where I must profitably put my time.

I have however, notified my customers that I am giving notice on all BPI 
cryopreservation contract services.  This is being done because I wish to 
add some language relating to media and precryopreservation care, and 
because I do NOT intend to more human cases unless I can take small, 
clinically acceptable (by today's standards) brain biopsies from carefully 
selected areas of the nondominant hemisphere.  I am tired of not having 
feedback and there is no substitue for REAL patients, for HUMANS.  And 
there is no substitute for this feedback in achieving quality control.  
Brian alludes to this in his response; since he opened the door, I've 
walked through it.

Finally I note that Donalson in his CRYONICS article notes that monkeys can 
recover from long periods (15+ minutes) of global brain ischemia.  This is 
true and this WHY we don't use them.  Dogs are MORE like people than 
monkeys; especially monkeys that swing from trees.  It's a wild guess but 
I'd bet that brain protection and repair systems are upregulated in monkeys 
(this could be a useful area for study in and of itself!).  Whatever the 
reasons, the outcome, the histology and the ultrastructure of monkey brains 
in resoponse to ischemia DO NOT map what happens in humans well at all.  
Dogs do: and this is why use them.  Cats seem to be intermediate between 
the two.

As to the notion that humans are being salvaged over comparable periods of 
time this is nonsense.  Yes, you can find anecdotal reports, but there are 
no RCTs that I know of or even large-scale non RCT studies.  Donaldson 
remarks that: "By now some neurologists have learned to use several drugs 
(nimodipine, naloxone, pentobarbital)
to lessen these effects (sic) of brain ischemia.

I  would like to see the hard data here.  I personally saw the early RCTs 
underway with Naloxone by Toth at Methodist hospital in 1979-80.  It didn't 
work.  Recent large studies on Nimodipine in the clinical setting (i.e., 
not in the lab) showed DECREASED survival and increased morbidity, probably 
due to the negative impact of the drug on blood pressure: if there's one 
thing Hossman and Safar have taught us, its the importance of a 
hypertensive bout following the ischemic interval (in part to open up 
capillaries full of hyperviscous blood and ATP depleted and thus rigid RBC 
and WBCs (water quickly gets extracted from the boood and absorbed by the 
swelling glial cells and neurons and this increases the colloid 
concentration of the capillary blood enormously making it thick and 
resistant to reflow).  One reason that this hypertensive bout has not been 
applied clinicaly is that hearts STOPPED by heart-attack and subjected to 
ischemia while fibrillating (using about 300 times their resting engery 
requirements!) don't easily deliver mean arterial pressures (MAPS) of 150 
mmhg!!!!!

As to pentobarbital; it is no longer widely used.  In our hands it has had 
NO protective effect given before or after ischemia of more than 10 
minutes.  Given before in shorter episodes it is modestly protective in 
very high doses.  Given after, not protective in terms of outcome 
(neurological recovery) as far as we can see.  It also depresses blood 
pressure which is definitely the kiss of death in and of itself.

A long answer, and an answer to some questions not asked :).  But I make no 
apologies.

Mike Darwin


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